ABSTRACT Notch signaling in alloimmunity Allogeneic T cell responses against foreign host antigens mediate graft-versus-host disease, the most serious complication of allogeneic bone marrow transplantation (allo-BMT). During the first five years of this proposal, we defined a new critical role of Notch signaling in alloreactive T cells mediating graft-versus-host disease after allo-BMT. Inhibition of canonical Notch signaling in donor T cells markedly reduced the severity and mortality of graft-versus-host disease in multiple mouse models of allo-BMT. Notch blockade induced a unique pattern of immunomodulation in T cells, with decreased production of inflammatory cytokines and increased expansion or regulatory T cells. However, in vivo T cell proliferation, expansion in lympho-hematopoietic organs and cytotoxicity were preserved upon Notch inhibition. Notch1/2 receptors in T cells and Delta-like1/4 (Dll1/4) Notch ligands in the host played a dominant role. Dll1/4 inhibition emerged as a promising strategy to inhibit Notch signaling without inducing side effects from systemic pan-Notch inhibition. Interestingly, we discovered that short-term Dll1/4 inhibition in the peri-transplant period was sufficient to confer long-term protection from morbidity and mortality. Dll1/4 blockade during the first 48 hours after allo-HCT was essential to efficiently control graft-versus-host disease, suggesting that a critical early pulse of Notch signaling is delivered to incoming donor T cells, with a long-lasting impact on their pathogenic functions. Furthermore, we identified specialized radioresistant stromal cells in secondary lymphoid organs as the critical source of Dll1/4 Notch ligands after lethal irradiation and allo-BMT. We hypothesize that alloantigen-specific T cells form early contacts with resident subsets of non-hematopoietic stromal cells in secondary lymphoid organs that drive T cell pathogenicity in graft-versus-host disease through Dll1/4-mediated Notch signals. To explore this hypothesis in detail, we will investigate the distribution of Dll1/4-expressing cells and test the importance of defined cellular sources of Notch ligands in multiple models of graft-versus-host disease driven by major or minor alloantigen mismatches, and with graded doses of irradiation; visualize and define the interaction of alloantigen- specific T cells with cellular sources of alloantigens and Notch ligands in secondary lymphoid organs; and explore the cellular and molecular mechanisms of Notch action in alloreactive T cells during their initial window of Notch sensitivity in vivo. These studies will bring novel insights into the molecular regulation of alloimmunity and might lead to the development of new approaches to limit damaging consequences of T cell reactivity after allogeneic transplantation.